1. Gene 210 Fetal/Prenatal Sequencing May 19, 2015

2. Today’s Plan Innovations in prenatal diagnosis (Gitler) Anneuploidy Mendelian disorders Non-invasive diagnostic technologies Yair Blumenfeld, M.D. clinical aspects cell-free RNA and non-aneuploidy issues

3. Prenatal Diagnosis Fetal aneuploidy and other chromosomal defects affect 0.9% of live births 3,000 rare genetic disorders, which collectively are not rare (1%)

4. Pre-implantation Genetic Testing during IVF Embryo biopsy of single blastomere at 6-8 cell stage Most common autosomal dominant and recessive diseases (e.g. cystic fibrosis, thalassemias, sickle cell anemia, and Duchenne muscular dystrophy, Huntington Disease) as well as chromosomal translocations

5. Human chromosomal aneuploidy syndromes

6. Trisomy 21 (Down Syndrome) Most common chromosome abnormality in humans. Why? ~1 out of 691 births in US Increased incidence of Alzheimer disease Increased incidence of leukemia but decreased incidence of most other cancer types Increased incidence of congenital heart disease

7. Trisomy 18 (Edwards Syndrome) ~1 out of 6,000 births in US ~80% females Very low survival rate (median lifespan 5-15 days) 8% survive < 1 year 1% survive to age 10 Major heart, kidney, and other organ abnormalities

8. Trisomy 13 (Patau syndrome) ~1 out of 10,000 births in US Multiple organ defects Mental retardation and motor impairment Holoprosencephaly Polydactyly >80% of children born with trisomy 13 die within 1 st year of life

9. Prenatal Diagnosis Non-invasive Increased fetal nuchal translucency) 75-80% sensitivity, 5-6% false positive rate Performed 11-14 weeks of gestation Ultrasound results combined with additional non-invasive tests: free beta-hCG/PAPP-A screen (85% sensitivity) alpha-fetoprotein, unconjugated estriol, beta-hCG, and inhibin- Alpha (INHA) (15-20 weeks) (81% sensitivity) Combined: 95% sensitivity, 5% false positive

10. Prenatal Diagnosis Invasive Amniocentesis: A sample of the amniotic fluid surrounding the fetus is withdrawn through a needle inserted into the mother's uterus. Fetal chromosomes are analyzed. After 15 weeks of gestation. 1:200 risk of miscarriage. Chorionic villus sampling (CVS): Cells taken from the embryo placenta can be used to analyze the fetal chromosomes. Typically performed between week 9 and 14. 1:100 risk of miscarriage.

11. Presence of fetal DNA in maternal plasma and serum Dennis Lo et al., Lancet 1997 Fetus-derived Y sequences were detected in 24 (80%) of the 30 maternal plasma samples, and in 21 (70%) of the 30 maternal serum samples, from women bearing male fetuses. These results were obtained with only 10 μL of the samples. None of the 13 women bearing female fetuses, and none of the ten non-pregnant control women, had positive results for plasma, serum or nucleated blood cells. “Our finding of circulating fetal DNA in maternal plasma may have implications for non-invasive prenatal diagnosis…”

12. Biology of circulating fetal DNA Consists predominantly of short DNA fragments (80% <200bp) Small fraction (3–6%) of cell-free DNA present in maternal plasma Earliest gestational age detectable is day 18 after embryo transfer by assisted reproduction (i.e., IVF) Fetal DNA concentration increases with gestational age Rapidly cleared following delivery (mean half-life 16 minutes) Placenta is predominant source of fetal DNA in maternal plasma

13. Lo et al., Nat Rev Genet 2007

17. Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18

20. Establishing the copy number of fetal chromosomes through analysis of allele ratios in the maternal plasma

25. David Ferreiro, an investment banker at Oppenheimer based in New York, has estimated that the non-invasive prenatal-testing market could exceed $1 billion a year. So far, sales volumes reported by Sequenom — the only publicly traded company of the four — have “substantially exceeded expectations”, says Ferreiro.

26. 14 October 2005 Sequenom licenses a patent for non-invasive prenatal diagnosis. 10 August 2010 Sequenom’s lawyers send Verinata Health a letter warning that Verinata is developing tests that will infringe on Sequenom’s patent and patent application. 30 August 2011 Stephen Quake — founder of Verinata — and Hei-Mun Fan, both at Stanford University in California, are issued a patent for ‘Determination of fetal aneuploidies by massively parallel DNA sequencing’. 17 October 2011 Sequenom launches the MaterniT21 test. 6 December 2011 Sequenom sends Aria Diagnostics a letter warning about patent infringement. 19 December 2011 Aria files a complaint against Sequenom. 6 January 2012 Natera files a complaint against Sequenom. 24 January 2012 Sequenom sues Aria. 22 February 2012 Verinata and Stanford University sue Sequenom. 1 March 2012 Verinata launches the Verifi prenatal test. 7 May 2012 Ariosa (formerly Aria) launches the Harmony prenatal test. Blood Feuds Nature 2012

31. There were 9 false positives among the 15,803 women in the cfDNA-testing group without trisomy 21, for a false positive rate of 0.06% (95% CI, 0.03 to 0.11) There were 854 false positive results for trisomy 21 on standard screening, for a false positive rate of 5.4% (95% CI, 5.1 to 5.8; P<0.001) The positive predictive value was 80.9% (95% CI, 66.7 to 90.9) for cfDNA testing and 3.4% (95% CI, 2.3 to 4.8) for standard screening (P<0.001)

33. Prenatal diagnosis of  thalassemia by sequencing from maternal plasma

35. Bianchi Nature 2012

36. Inference of the fetal genome on a site-by-site basis

37. Inference of the fetal genome from haplotype blocks

38. De novo missense mutation in the gene ACMSD detected

40. Molecular counting strategies for measuring the fetal genome non-invasively from maternal blood only

41. Prenatal diagnosis of DiGeorge Syndrome

42. a longitudinal study on pregnant women and analyzed their combined cell- free RNA transcriptomes across all three trimesters of pregnancy and after delivery. possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer’s disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals.

43. Non-Invasive Fetal Genome Sequencing: Opportunities and Challenges Advantages Earlier detection of genetic disorders allows for preparation for postnatal interventions (e.g., SCNA1 mutations cause seizures; spontaneous mutations causing multiple endocrine neoplasia --> prophylactic thyroidectomy)

44. Non-Invasive Fetal Genome Sequencing: Opportunities and Challenges Might increase rates of elective pregnancy termination. But could make that safer, if performed early in gestation. Societal pressure to terminate any fetus suspected to have a Mendelian condition?

45. Non-Invasive Fetal Genome Sequencing: Opportunities and Challenges If access to genetic testing limited by cost, might result in children with Mendelian conditions disproportionately born to lower income families What about variants identified beyond scope of study? E.g., variants that increase risk for a trait (How might this help parent?)

46. Non-Invasive Fetal Genome Sequencing: Opportunities and Challenges The ACMSD de novo mutation identified by Kitzman et al – is pregnancy more vulnerable time to make decision – more weight given to information than warranted? Does this information go in baby’s medical record? Updated and continually mined? Or destroyed? Who controls it?

47. Today’s Plan Innovations in prenatal diagnosis (Gitler) Anneuploidy Mendelian disorders Non-invasive diagnostic technologies Yair Blumenfeld, M.D. clinical aspects cell-free RNA and non-aneuploidy issues